The present invention relates generally to materials and methods for use in immunological procedures for isolation and quantitative detection of hematopoietic growth factors from biological fluids. Specifically, the invention relates to monoclonal antibodies produced by novel hybridoma cell lines (as exemplified by A.T.C.C. HB-8957, A.T.C.C. HB-8958, A.T.C.C. HB-8959, A.T.C.C. HB-8960, A.T.C.C. HB-8961 and A.T.C.C. HB-8962) which are specifically reactive with hematopoietic growth factors and to uses of these antibodies in isolation of hematopoietic growth factors through affinity purification techniques, in assays for detection of hematopoietic growth factors and in immunological techniques for study of such growth factors. More specifically, the invention relates to monoclonal antibodies specifically reactive with human pluripotent granulocyte colony stimulating factor (hpG-CSF) from natural and recombinant sources.
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic system of the average human male has been estimated to produce on the order of 4.5.times.10.sup.11 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight. Dexter, et al., BioEssays, 2, 154-158 (1985).
It is believed that small amounts of certain hematopoietic growth factors account for the differentiation of a small number of progenitor "stem cells" into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the ultimate differentiation of mature blood cells from those lines. Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions. As a result, a large number of names have been coined to denote a much smaller number of factors. For example of the resultant confusion the terms, IL-3, BPA, multi-CSF, HCGF, MCGF and PSF are all acronyms which are now believed to apply to a single murine hematopoietic growth factor. Metcalf, Science, 229, 16-22 (1985).
The application of recombinant genetic techniques has brought some order out of this chaos. For example, the amino acid and DNA sequences for human erythropoietin, which stimulates the production of erythrocytes, have been obtained. (See, Lin, PCT Published Application No. 85/02610, published Jun. 20, 1985.) Recombinant methods have also been applied to the isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human macrophage-specific colony-stimulating factor (CSF-1). [See, Lee, et al., Proc. Natl. Acad. Sci. (USA), 82, 4360-4364 (1985); Wong, et al., Science, 228, 810-814 (1985) and Kowasaki, et al., Science, 230, 291-296 (1985).]
A human hematopoietic growth factor, called human pluripotent colony-stimulating factor (hpCSF) or pluripoietin, has been shown to be present in the culture medium of a human bladder carcinoma cell line denominated 5637 and deposited under restrictive conditions with the American Type Culture Collection, Rockville, Md. as A.T.C.C. Deposit No. HTB-9. The hpCSF purified from this cell line has been reported to stimulate proliferation and differentiation of pluripotent progenitor cells leading to the production of all major blood cell types in assays using human bone marrow progenitor cells. Welte, et al., Proc. Natl. Acad. Sci. (USA), 82, 1526-1530 (1985).
Preliminary studies indicate that the factor identified as hpCSF has predominately granulocyte colony-stimulating activity during the first seven days in a human CFU-GM assay.
Another factor, designated human CSF-.beta. has also been isolated from human bladder carcinoma cell line 5637 and has been described as a competitor of murine .sup.125 I-labelled granulocyte colony-stimulating factor (G-CSF) for binding to WEHI-3B D.sup.+ cells in a dose-response relationship identical to that of unlabelled murine G-CSF [Nicola, et al., Nature, 314, 625-628 (1985)]. This dose-response relationship had previously been reported to be unique to unlabelled murine G-CSF and not possessed by such factors as M-CSF, GM-CSF, Il-3 [Nicola, et al., Proc. Natl. Acad. Sci. (USA), 81, 3765-3769 (1984)]. See also, Metcalf, et al., Leukemia Research, Vol. 9, No. 5, pp. 521-527 (1985). CSF-.beta. and G-CSF are also unique among CSF's in that they share a high degree of ability to induce differentiation of WEHI-3B D.sup.+ cells. Nicola, et al., Immunology Today, 5, 76-80 (1984). At high concentrations, G-CSF stimulates mixed granulocyte-macrophage colony-forming cells [Nicola, et al., (1984) supra], which is consistent with preliminary results indicating the appearance of granulocytic, monocytic, mixed granulocytic/monocytic and eosinophilic colonies (CFU-GEMM) after 14 days incubation of human bone marrow cultures with hpCSF. CSF-.beta. has also been described as stimulating formation of neutrophilic granulocytic colonies in assays which employed mouse bone marrow cells, a property which has been a criterion for identification of a factor as a G-CSF. On the basis of these similarities, human CSF-.beta. has been identified with G-CSF (granulocytic colony stimulating factor). See, Nicola, et al., Nature, 314, 625-628 (1985).
Based upon their common properties, it appears that human CSF-.beta. of Nicola, et al., supra, and the hpCSF of Welte, et al., supra, are the same factor which could properly be referred to as a human pluripotent granulocyte colony-stimulating factor (hpG-CSF).
Souza U.S. Pat. No. 4,810,643, hereby incorporated by reference, novel recombinant-produced polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties of human pluripotent granulocyte colony stimulating factor are disclosed. Also disclosed are DNA sequences coding for such polypeptides, transformed host cells coding for such polypeptides and processes for the synthesis of such factors by recombinant methods.
Of interest to the background of the invention is current research focused on hybridoma techniques for producing tumor cell lines which will manufacture highly specific monoclonal antibodies to a selected antigenic substance. Techniques for the production of monoclonal antibodies are generally well known in the art. Typical descriptions of these procedures may be found in Wands, J. R., and Zurawski, V. R., Gastroenterology 80:225 (1981); Marshak-Rothstein, et al., J. Immunol. 122:2491 (1979); and Oi, V. T. and L. A. Herzenberg, "Immunoglobulin Producing Hybrid," Mishell, B. B. and S. M. Shiigi (eds.) Selected Methods in Cellular Immunology, San Francisco: W. H. Freeman Publishing, 1979 and Goding, "Antibody Production by Hybridomas" J. of Immunol. Meth. 39, 285-308 (1980). Briefly summarized, lymphocytes removed from the spleen of an animal previously injected with the antigen of interest are induced to fuse with myeloma cells in the presence of polyethylene glycol. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones are individually assayed for supernatant activity.
Due to the highly specific nature of their immunological properties, monoclonal antibodies developed according to hybridoma techniques have been proposed for use as diagnostic reagents, therapeutic agents, and agents for affinity purification of specifically cross-reactive antigenic proteins from crude sources. See, e.g., Trends in Biotechnology, Vol. 3, No. 7 (July, 1985) and U.S. Pat. Nos. 4,465,624, 4,514,505 and 4,514,507.
While there exists a substantial need for specific monoclonal antibodies for use in detecting, isolating, purifying and studying hematopoietic growth factors such as human pluripotent granulocyte colony-stimulating factor, there have been no reports of the successful use of hybridoma techniques in obtaining monoclonal antibodies to hpG-CSF.